High-pH Reversed-Phase Fractionation and SPS-MS3 TMT Data Acquisition

Fractionation and TMT data acquisition were performed as previously described (Mervosh et al., 2018). Briefly, high-pH reversed-phase C18 peptide fractionation was performed on an ACQUITY UPLC H-class system (Waters Corporation, Milford, MA, United States) equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm). Elution was performed as previously described (Mervosh et al., 2018), with a flowrate of 0.3 ml/min and gradient of 2% of mobile phase A (10 mM ammonium acetate, pH 10) to 37% mobile phase B (10 mM ammonium acetate in 90% acetonitrile, pH 10) in 19.8 min. In total 60 fractions were collected, pooled into five fractions, dried, and reconstituted in loading buffer (0.2% trifluoroacetic acid, 2% acetonitrile in water).

Reversed phase-liquid chromatography-mass spectroscopy (RP-LC-MS/MS/MS) was performed at the Yale/Keck MS & Proteomics Resource with a nanoACQUITY UPLC system (Waters Corporation, Milford, MA, United States) connected to an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, United States) as described (Mervosh et al., 2018). SPS-MS3 scanning was performed as described previously (Mervosh et al., 2018), except that the maximum injection time was set to 60 ms, and dynamic exclusion was enabled for a duration of 30 s for the full scan. CID-MS fragmentation isolation mode was set to quadrupole and isolation width was set to 1.6 m/z. MS3 scans were produced with higher-energy collision dissociation (HCD) for the top 10 fragment ions for each peptide MS2 and were analyzed in the Orbitrap at a resolution of 60,000. The maximum injection time was set to 120 ms and automatic gain control (AGC) target value was set to 1 × 10⁵.